PROJECT SUMMARY/ABSTRACT Atherosclerosis is a chronic inflammatory disorder that affects approximately 70% of older men and women ?65 years old, causing heart attack, stroke, heart failure, limb loss, dementia, and even death. Older adults may be more susceptible to atherosclerosis and its adverse sequelae because of immune senescence, defined as the gradual deterioration of the immune system due to natural aging. While many studies have shown that pro-inflammatory T cells detected in the blood increases the risk of plaque progression and rupture, little is known about how antigen specific T cell activation and/or T cell immune senescence in the atherosclerotic plaque alters the function of T cell subsets and promotes a more vulnerable phenotype. There is a critical need to better understand the mechanisms by which T cell senescence contributes to the pathogenesis of atherosclerosis; without this information, pharmacotherapy designed to mitigate the effects of an aging immune system on atherosclerosis and its adverse sequelae will be unattainable. Because of the development of single cell technology and next generation sequencing that is specifically designed to better understand T cell biology by the applicant's mentor, the fundamental question of whether T cell clonality and senescence contributes to the development of atherosclerosis and its adverse sequelae in older adults can now be answered. Over the past year, the applicant has applied these techniques to begin answering this question and has found the presence of clonal, immunosenescent T cells in the atherosclerotic plaque in four older adults. The following plan will address this problem: Aim 1: Validate clonal T cell populations that cause atherosclerosis in older adults. In this aim, T cell receptors (TCRs) at the single cell level will be sequenced and phenotyped using an approach pioneered by the applicant's mentor and already successfully performed by the applicant; Aim 2: Identify antigen(s) that activate oligoclonal T cell expansion in atherosclerotic plaque isolated from older patients with advanced atherosclerosis. TCR data generated in Aim 1 will be organized and analyzed for antigen identification by a well-validated algorithm developed by the applicant's mentor; and Aim 3: Characterize antigen-specific T cells that distinguish unstable from stable disease. Antigen specific T cells will be characterized using novel peptide-MHC dodecamer technology developed by the applicant's mentor. This proposal is innovative because it departs from the status quo by utilizing advances in single cell sequencing, computational analysis, and dodecamer technology to enable the development of immune based therapy that directly targets plaque progression and rupture, opening new research horizons. The proposed research is significant, because it is expected to vertically advance and expand our understanding of how antigen specific T cell activation and/or immune senescence alters T cell function and promotes plaque progression and rupture. Such knowledge will enable the development of immune-based therapies to target atherosclerosis in older adults.